The signaling protein Galpha-i2, a member of the G-protein family of signal transduction proteins, mimics insulin signaling, and appears to play important role in cell differentiation because changes in its cellular levels seem to correlate with either inhibition or induction of cell differentiation, depending on the cell type. Its expression is altered in diverse situatiom including neonatal development in liver and differentiation in hematopoietic cells in culture, implying functional association between expressed levels of this protein and several biological processes. Presently, it is unclear how changes in Galpha-i2 levels determine the extent of cell differentiation; it is also unclear whether Galpha-i2, by itself, can trigger differentiation. This project has two general objectives: 1) to unravel fandamemal molecular mechanisms that regulate the cellular levels and expression of Galpha-i2; and 2) to explore the molecular basis for how Galpha-i2 influences cell differentiation. Specifically, the studies will explore signaling pathway(s) by which Galpha-i2 influences cell differentiation and will use the yeast two-hybrid system to identify putative direct interacting partner(s) that is/are relevant to transmission of Galpha-i2-initiated signals that impact cell differentiation. Using differentiation as a means to engender changes in expression of Galpha-i2, transcription factors that mediate this expression will be identified. Interactions between identified transcription factors and potential transcriptional accessory factors will also be studied. Rates of synthesis and degradation of Galpha-i2 during cell differentiation will be measured as well as transcription rates for Galpha-i2, and the stability of its mRNA. These experiments will provide fundamental information about mechanisms that control cellular levels of this G protein. They will also provide information on the molecular basis for the Galpha-i2-dependent changes in cell differentiation. Such information may be applicable to the regulation of other G proteins. The studies proposed here will be carried out with K562 cells. KS62 cells are pluripotent hematopoietic ceils that can he made to differentiate along erythroblastic, monocytic or megakaryocytic lineage, depending on the cell differentiation inducer used, thus providing an excellent paradigm for testing functional correlates such as the ones to be studied in this project.